Project Summary Understanding the mechanisms of genetically-defined rare diseases can provide insight into disease pathogenesis. In humans, mutations in genes encoding mitochondrial proteins lead to a wide spectrum of diseases. A subset of these mutations, those in the biosynthesis pathway of Coenzyme Q10, manifest as an autosomal recessive inherited progressive encephalopathy and/or nephrotic syndrome (NS). NS, the loss of the kidney filtration barrier, can be either inherited or acquired. NS has an incidence of approximately 3 new cases per 100,000 adults each year and over 6,000 new pediatric cases each year. Currently, there are no targeted therapies to treat NSL the one exception is Coenzyme Q10 supplementation in CoenzymeQ10 deficiency. The mechanism of disease, for both NS and Coenzyme Q10 deficiency, is poorly understood. However, due to the role of mitochondria in respiration, it is hypothesized that mutations may lead to cellular damage from reactive oxygen species (ROS). Additionally, ion channels, specifically TRPC5 which may be regulated by ROS, have been implicated in NS pathogenesis, and our lab has shown that TRPC5 regulates the actin cytoskeleton in podocytes (cells critical to kidney filter function). Thus, I propose to investigate the role of human mitochondrial mutations in altering cellular redox balance and ion channel activity. The central hypothesis of this proposal is that human mitochondrial mutations in coenzyme Q10 biosynthesis cause podocyte injury through alterations in ion channel activity via oxidative stress. Based on human mitochondrial mutations, Coenzyme Q10 biosynthesis enzymes will be knocked out in cultured podocytes using a CRISPR/Cas9 system to assay the effect on mitochondrial function and redox balance. It will then be determined how these mutations affect the abundance, localization, and function of TRPC5 and the cytoskeleton. To complement this hypothesis-driven approach, an unbiased proteomics screen will be performed, using mitochondrial- or plasma-membrane-targeted APEX, to look more broadly for alterations in ion channel expression. Finally, mouse models will be made to introduce human mutations, based on the enzyme(s) best characterized in in vitro assays, and the effects on kidney function, oxidative stress, and ion channel activity will be characterized. This work will demonstrate mechanisms by which mitochondrial defects can directly affect podocyte cytoskeleton dynamics or podocyte survival, and thus reveal much needed therapeutic targets for NS.